Reliable sex determination is an inevitable prerequisite in prenatal and preimplantation diagnosis of X-linked diseases. We report on an amelogenin-based nested polymerase chain reaction sexing method that simultaneously amplifies distinguishable fragments from both sex chromosomes. Primers matching a largely homologous region on both sex chromosomes are used that encompass a 177-bp deletion on the Y chromosome. Thus amplification results in X- and Y-specific fragments of different sizes that are resolved simply by agarose gel electrophoresis. We applied our sexing strategy to 102 single amniocytes previously subjected to primer extension preamplification. 95 showed successful amplification (93.14 sensitivity). The genotyping of all successful amplifications (from 42 male and 53 female amniocytes) was found to be correct (100 specificity). None of the media blanks showed amplification products (no false positives). Additional amplification of the locus of the most common cystic fibrosis mutation resulted in 95.1 success: 89 amniocytes (87.3) showed no mutated allele and 7 (6.9) were found to be heterozygous for the ΔF508 mutation
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